EP1094899B1 - Method for separation on cation exchangers - Google Patents

Method for separation on cation exchangers Download PDF

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Publication number
EP1094899B1
EP1094899B1 EP99931714A EP99931714A EP1094899B1 EP 1094899 B1 EP1094899 B1 EP 1094899B1 EP 99931714 A EP99931714 A EP 99931714A EP 99931714 A EP99931714 A EP 99931714A EP 1094899 B1 EP1094899 B1 EP 1094899B1
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EP
European Patent Office
Prior art keywords
group
groups
cation
ionic strength
cation exchanger
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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EP99931714A
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German (de)
English (en)
French (fr)
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EP1094899A1 (en
Inventor
Rolf Berglund
Jan Bergström
Lennart SÖDERBERG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Sweden AB
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Amersham Bioscience AB
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Publication of EP1094899A1 publication Critical patent/EP1094899A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/04—Processes using organic exchangers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/16—Organic material
    • B01J39/18—Macromolecular compounds
    • B01J39/19—Macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds

Definitions

  • protein comprises compounds containing peptide bonds between amino acids.
  • the term comprises oligopeptides and polypeptides as well as lipoproteins, glycoproteins, proteoglycans, etc.
  • the matrix may be based on inorganic compounds, synthetic resins, polysaccharides, etc. Different matrices may have different physical properties. Porosity, mechanical strength, rigidity, flow characteristics, swelling properties, the degree of non-specific adsorption, etc of the matrices may vary.
  • Separation by cation exchange to purify a substance is often combined with other techniques distinguishing compounds based on differences in size and form (gel filtration), in biospecific affinity (bioaffinity chromatography), in ability for hydrophobic interaction etc.
  • Purification of a compound is usually initiated by extraction.
  • the resultant crude extract typically has a large volume and such a high ionic strength that it is not possible to adsorb on conventional ion exchangers without dilution, which further will increase the volume (conventional cation exchangers don't have sufficient adsorption ability at a high ionic strength). Large volumes will thus have to be handled and therefore relevant investments must be made in space demanding and expensive equipment and also use of highly purified water.
  • EP 326233 discloses a cation exchanger in which there is a hydrophobic base matrix to which cation exchanging groups are attached.
  • the hydrophobicity of this type of cation exchangers makes them unsuitable for separation of biomolecules such as proteins.
  • WO 9808603 discloses separation media of the general structure M-SP1-L in which M is a base matrix, SP1 is a spacer and L is a ligand comprising a mono- or bicyclic aromatic or heteroaromatic moiety that may substituted.
  • L is X-A-SUB where X is -O-, -S- or -NH- and A the aromatic or heteroaromatic moiety that is substituted.
  • the substituent on A may be an acidic group.
  • the separation media are suggested for the isolation of proteins, in particular immunoglobulins. The publication does not disclose the properties we have discovered and contains no disclosure of a separation method utilizing these properties.
  • US 5,652,348 discloses ion exchange resins in which the hydrophobicity/hydrophilicity of the resin including the ligand is changed upon change in pH.
  • the hydrophobicity may be increased by the introduction of hydrophobic non-ionizable ligands.
  • Adsorption/desorption is controlled by altering the hydrophobicity/hydrophilicity of the matrix including ligand.
  • the instant invention is based on our discovery that cation exchangers carrying ligands of a certain kind of structure may exhibit unexpected strong interactions with positively charged compounds of complex structure, for instance macromolecules such as proteins and many other biomolecules. This is reflected in our finding that this kind of cation exchangers often require an abnormally high ionic strength for elution of adsorbed proteins.
  • Cyclopentyl and cyclohexyl are the most important alicyclic groups that can be used as Y.
  • alicyclic, aliphatic and aromatic structures may be substituted by 1, 2, 3, 4, 5 or 6 OR groups.
  • RO is OH or lower alkoxy or any other alkoxy group not disturbing the advantageous properties utilized in the invention.
  • lower alkoxy and lower alkyl are meant C 1-10 , preferably C 1-6 alk-oxy/alkyl.
  • R'O groups can be placed in Y, in Ri or R 2 or in the spacer A.
  • R'O is preferably OH.
  • Y is an aliphatic group it is preferably a C 2 -C 8 group, such as a C 2 -C 6 group, preferably a C 2 -C 4 group, which groups may be substituted by OR groups according to above.
  • Y a C 1 group
  • X -N(R 1 )(R 2 )-, where one of R 1 or R 2 is a free electron pair or hydrogen and the other is an alkyl according to above with 1, 2 or 3 OR at a distance of two carbon atoms from N with RO being as defined above.
  • the cation exchangers to be used in the invention exhibit a higher, preferably more than 25% higher, such as more than 40% and even more than 100% higher maximum elution ionic strength in the pH interval 2-12 for one or more proteins selected among ribonclease, chymotrypsinogen A, cytochrome C, lysozyme, wheat-germ lectin and ⁇ -lactoglobulin, compared with the maximum elution ion strength required for the same proteins on a reference ion exchanger where the ion exchanging group is sulphonate (-SO 3 - ) and the base matrix, the spacer and the degree of substitution are the same but where the groups X and Y are absent.
  • the most important aspect of the present invention is a process for separation of positively charged compounds from a solutions according to claim 1.
  • the process comprises that the solution is contacted with a cation exchanger to bind one or more of the positively charged compounds to the cation exchanger, and also, if desired, to elute/desorb bound compounds.
  • the compounds concerned are primarily biomolecules and particularly amphoteric ones, such as proteins.
  • the characterizing features of the process is (a) that the cation exchanger is according to Formula (I) and have higher maximum elution ionic strength in relation to a reference sulphonate cation exchanger in the same manner as desrcribed above, and (b) that the binding between the cation exchanger and a positively charged compound is allowed to occur at a pH value within the interval 2-14 where a significant part of the ligands (-X-Y(-Z) n ) have a negative net charge, preferentially all of them.
  • this kind of cation exchangers means that the binding often may occur at a higher ionic strength than the elution ionic strength required when eluting the compound at the same pH value from a reference ion exchanger according to above. The comparison is made with pH and other conditions being the same.
  • the cation exchanger of the invention may be selected so that the ionic strength, when binding occurs, may be at least 25% higher, such as at least 40% higher than when using the corresponding reference ion exchanger according to above (as measured at the same pH). If required binding may also be performed at more than 100% higher ionic strength than when using the corresponding reference ion exchanger according to above (as measured at the same pH ).
  • this may involve the binding being performed at an ionic strength exceeding 15 or 20 mS/cm, such as exceeding 30 mS/cm and in some cases exceeding 40 mS/cm.
  • the applicable figures in a particular case will depend on the selection of ligand and compound to be eluted.
  • An interesting application of this embodiment of the invention is large-scale processes in which large volumes of a crude product having a high ionic strength are to be applied on the cation exchanger.
  • diluting is required in order to enable binding of the compound of interest to the conventional ion exchanger.
  • the need of diluting is often reduced.
  • the elution step in the process of the invention may, for amphoteric compounds and other compounds that may be positively charged, such as proteins, primarily be performed according to four main alternatives
  • any condition/methodology alone or in combination that neutralizes the interaction causing binding may be utilized for desorption.
  • the ligand analogue may be combined with any one of alternatives a-c.
  • the most advantageous alternatives typically neutralize the interaction causing binding without requiring an increase in the ionic strength.
  • the eluate will contain the released substance and will have a lowered salt concentration compared to the sample originally applied. The requirements of desalting may thus be reduced.
  • elution by changing the pH of the eluting liquid (alternatives a) combined with a lowering of ionic strength is particularly preferred among alternatives (a)-(c).
  • the alternative with a ligand analogue may give similar advantages.
  • elution/desorption of the ion exchanger usually has to be performed at an ionic strength which is higher than the corresponding elution ionic strength required for a corresponding reference ion exchanger according to above (as measured at the same pH).
  • the ionic strength in this step may thus be at least 25% higher such as at least 40% higher than for the reference cation exchanger. In some extreme cases an ionic strength may be required that is more than 100% higher than for the corresponding reference ion exchanger (as measured at the same pH).
  • Another preferred embodiment involves selecting the cation exchanger according to Formula I so that elution can be performed by use of such a ionic strength gradient that the elution interval becomes broader than the interval required at the same pH value for the reference ion exchanger according to above.
  • This will normally be accomplished by selecting the cation exchanger of formula I according to the preferred modes described above.
  • the interval referred to may start at the lowest ionic strength applied for elution and ends at the ionic strength at the maximal peak height for a compound of interest.
  • the interval may be the interval defined by elution ionic strength for peak maximum for two different compounds appearing during elution. This means greater possibilities to separate compounds, which are related to each other with reference to charge, and/or to improve yield of these compounds.
  • the matrix is often porous.
  • the spacer A joining the ion exchange ligand -X-Y(-Z) n with the matrix M in the cation exchanger, may be of the same type as in traditional ion exchangers.
  • the demands for hydrolytic stability require that spacers should be built of groups that are stable against hydrolysis. They may for instance contain groups selected among straight, branched, cyclic saturated and unsaturated and aromatic hydrocarbon groups (e.g.
  • the spacer binds to X via a sp 3 -hybridized or aromatic carbon atom.
  • the brominated gel was loaded together with 30 ml of distilled water and 8 g of sodium sulphite into a three-necked 100 ml Bellco flask with a hanging magnetic stirrer. PH was adjusted to about 10-11. The reaction was proceeded over night (23 hours) at 50°C.
  • the reaction mixture was neutralized with acetic acid.
  • the gel was then washed with distilled water (> 10 bed volumes) and for possible storage distilled water containing 23% (w/w) ethanol was used.
  • the gel was suctioned dried and loaded together with 30 ml of distilled water, 43.1 g (0.2476 mole) of dipotassium hydrogen phosphate and 9.90 g of sodium hydroxide into a three-necked 25 ml Bellco flask with a hanging magnetic stirrer. The reaction was proceeded over night 16-22 hours at 40°C. The reaction was stopped by lowering the pH to 7.0 with conc. HCl and then the gel was washed on a glass filter with distilled water (> 10 bed volumes).
  • the reaction was stopped by taking up the gel on a glass filter funnel and washed with a few bed volumes distilled water and then the gel was suspended in distilled water and the pH value was adjusted with acetic acid to about 6.
  • the gel was suctioned dried for 15-30 seconds and then loaded into a solution of 19.26 g (0.112 mole) of 4-sulphanilic acid and 7.34 g (» 0.1112 mole) of potassium hydroxide (>85%) in 20 ml of distilled water. First lye and water were mixed. The reaction was run in a three-necked 25 ml Bellco flask with a hanging magnetic stirrer. The reaction was performed during 16-22 hours at 40°C. The reaction was stopped by taking up the gel on a glass filter funnel and washed with a few bed volumes of distilled water and then the gel was suspended in distilled water and the pH value was adjusted with 1 M HCl to pH about 6.
  • a level of ligand of 0.12 mmole/ml packed gel was measured by adsorbing TRIS on gel packed in a column at pH 6, whereupon the excess was washed off and adsorbed TRIS was eluted with a sodium chloride solution.
  • the amount of TRIS in the eluate being equivalent to the amount of negative groups on the gel, was determined by inflectionpoint titration with sodium hydroxide.
  • the gel was suctioned dried for 15-30 seconds and then loaded into a solution of 56.54 g (0.2476 mole) of dihydroxybenzene sulphonic acid-potassium salt and 13.9 g of potassium hydroxide (> 85%) in 30 ml of distilled water and 0.1 g of sodium boron hydride.
  • the reaction was run in a three-necked 100 ml Bellco flask with a hanging magnetic stirrer. The reaction was proceeded over night, 16-22 hours, at 40°C.
  • the reaction was stopped by taking up the gel on a glass filter funnel and washed with a few bed volumes of distilled water and then the gel was suspended in distilled water and the pH value was adjusted with 1 M acetic acid to pH about 6.
  • the gel was suctioned dried for 15-30 seconds and loaded together with 30 ml of distilled water, 10 g (0.25 mole) sodium hydroxide and 60.25 g (0.24765 mole) of N-[Tris(hydroxymethyl)methyl]- 3-amino-propane-sulphonic acid into a three-necked 100 ml Bellco flask with a hanging magnetic stirrer. The reaction was performed over night, 16-22 hours, at 40°C.
  • the reaction was stopped by lowering the pH to 7.0 with conc. HCI, and then the gel was washed with distilled water (> 10 bed volumes).
  • Figure 1 Elution ionic strengths - comparison of different cation exchange ligands at pH 8.
  • Figure 2 Elution ionic strengths - comparison of different cation exchange lig ands at pH 4.
  • Weight indications (g) relate to vacuum drained gel on a glass filter funnel.
  • Ion exchange gels according to the descriptions for synthesis above were packed in a HR 10/2 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) to a gel height of 2-2.5 cm at back pressure of about 1 bar.
  • Figure 1 depicts the elution ionic strength in mS/cm at pH 8.0 for prepared ion exchangers for the studied model proteins.
  • Ion exchangers which under existing conditions exhibit a ligand with a negative net charge and an available functional group according to the invention have a different ability to separate the studied model proteins from each other by cation exchange chromatography.
  • One difference is a significant increased dynamics, i.e. the elution ionic strength values for the sample substances included in the separation extend over a wider ionic strength interval.
  • Another difference is that higher or considerably very much higher elution ionic strengths are required to elute all the used sample substances. These effects appear more or less simultaneously.
  • FIG. 2 shows results from several chromatography runs at pH 4. At this low pH value the ligands of the example have no or reduced negative net charge.
  • the TAPS-ligand is an example of a ligand where the net charge is zero and to which none of the sample substances adhere.
  • the sulphanilic acid group has a slightly positive net charge at pH 4.
  • the amino group is completely charged (pKa just above 7) and the sulphonate group (pKa of approximately 3) starts to uncharge.
  • the result for the partially uncharged sulphonate group in the sulphanilic acid-ligand illustrates that the interaction is complex and that the result maybe be difficult to interpret based on mechanistic terms.
  • the carboxylic group of the 4-hydroxybenzoic acid-ligand is also uncharged at pH 4, while the hydroquinone sulphonate-ligand is charged at pH 4 and gives strong binding.
  • Transforming a hydroxy group of the hydroquinone sulphate ligand to an ether group will dramatically change the binding capacity (for instance by (a) allylation and subsequent transformation of the double bound to a sulphonate group or (b) alkylation with BPR-butane (1,4-diglycidylbutylether)).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Treatment Of Water By Ion Exchange (AREA)
EP99931714A 1998-06-18 1999-06-16 Method for separation on cation exchangers Expired - Lifetime EP1094899B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9802214 1998-06-18
SE9802214A SE9802214D0 (sv) 1998-06-18 1998-06-18 Jonbytare och användning därav
PCT/SE1999/001082 WO1999065607A1 (en) 1998-06-18 1999-06-16 Method for separation on cation exchangers

Publications (2)

Publication Number Publication Date
EP1094899A1 EP1094899A1 (en) 2001-05-02
EP1094899B1 true EP1094899B1 (en) 2003-03-05

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EP99931714A Expired - Lifetime EP1094899B1 (en) 1998-06-18 1999-06-16 Method for separation on cation exchangers

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EP (1) EP1094899B1 (enExample)
JP (1) JP4643006B2 (enExample)
AT (1) ATE233599T1 (enExample)
AU (1) AU759380B2 (enExample)
CA (1) CA2334980C (enExample)
DE (1) DE69905720T2 (enExample)
SE (1) SE9802214D0 (enExample)
WO (1) WO1999065607A1 (enExample)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101363225B1 (ko) 2012-01-30 2014-02-17 (주)엔비엠 유전자 도입 벼 세포 배양액으로부터 재조합 인간 키모트립시노겐 ｂ２의 정제방법

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE9904197D0 (sv) * 1999-11-22 1999-11-22 Amersham Pharm Biotech Ab A method for anion exchange adsorption on matrices carrying mixed mode ligands
SE0002688D0 (sv) * 2000-07-17 2000-07-17 Amersham Pharm Biotech Ab Adsorption method and ligands
JP2004532182A (ja) * 2000-12-31 2004-10-21 アメルシャム・バイオサイエンシーズ・アクチボラグ 低濃度の塩を含有する組成物の製造方法
SE0004932D0 (sv) * 2000-12-31 2000-12-31 Apbiotech Ab A method for mixed mode adsorption and mixed mode adsorbents
SE0103084D0 (sv) * 2001-09-14 2001-09-14 Amersham Pharm Biotech Ab Generation of ion exchange media
SE526214C2 (sv) * 2003-02-28 2005-07-26 Amersham Biosciences Ab Ett sätt att generera metallkelaterande affinitetsligander
WO2008085116A1 (en) 2007-01-10 2008-07-17 Ge Healthcare Bio-Sciences Ab Mult i -modal ion exchange chromotography resins
JP5999899B2 (ja) 2008-04-08 2016-09-28 バイオ−ラッド ラボラトリーズ インコーポレーティッド 抗体のクロマトグラフィー精製法

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
GB8800169D0 (en) * 1988-01-06 1988-02-10 Dow Chemical Co Ion exchange resin & process for preparing
SE9600590D0 (sv) * 1996-02-19 1996-02-19 Pharmacia Biotech Ab Sätt för kromatografisk separation av peptider och nukleinsyra samt ny högaffin jonbytesmatris

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101363225B1 (ko) 2012-01-30 2014-02-17 (주)엔비엠 유전자 도입 벼 세포 배양액으로부터 재조합 인간 키모트립시노겐 ｂ２의 정제방법

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WO1999065607A1 (en) 1999-12-23
AU759380B2 (en) 2003-04-10
DE69905720T2 (de) 2004-01-15
DE69905720D1 (de) 2003-04-10
JP2002518157A (ja) 2002-06-25
JP4643006B2 (ja) 2011-03-02
AU4815099A (en) 2000-01-05
ATE233599T1 (de) 2003-03-15
CA2334980C (en) 2007-08-21
SE9802214D0 (sv) 1998-06-18
CA2334980A1 (en) 1999-12-23
EP1094899A1 (en) 2001-05-02

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